It has long been understood that morbidity and mortality caused by cancer, especially in view of the fact that surgical techniques are readily available, are due to more-or-less systemic sequelae of the original multiplication of the cancer cells. Metastasis of an original tumor to additional locations, the destruction of target locations as a direct or indirect result of this metastasis, cachexia, hypercalcemia, and other symptomologies characterize the course of the malignancy. The mechanisms whereby these sequelae occur are believed to involve a variety of cytokines, growth factors, and cell adhesion molecules, among other factors.
One such factor which is known to play a role in at least some of these processes is parathyroid hormone-related protein (PTH-rp). Due to its similarity to PTH, it has been recognized as a mediator of humoral hypercalcemia of malignancy (HHM). In this role, PTH-rp secreted by the tumor is circulated through the blood and is associated with hypercalcemia, an index of bone resorption.
PTH-rp has been purified from human lung cancer, breast cancer and renal cell carcinoma. Although PTH-rp was originally found associated with tumor cells, it is also present widely in normal tissue. The gene encoding PTH-rp was cloned and expressed by Suva, L. J. et al. Science (1987) 237:893-895. PTH-rp has been shown to bind to PTH receptors (Abou-Samra, A. et al. Proc Natl Acad Sci USA (1992) 89:2732-2736) and has activities similar to PTH (Horiuchi, N. et al. Science (1987) 23:1566-1568). It has been shown to stimulate adenylate cyclase in renal and bone systems, increase tubular resorption of calcium, decrease renal phosphate uptake and stimulate 1.alpha.-hydroxylase.
PTH-rp also has properties that are not shared by PTH, including regulation of placental calcium transport as summarized in a review by Mallette, L. E. Endocrine Rev (1991) 12:110-117. Of particular relevance with respect to the invention herein is that PTH-rp has also been associated with the establishment of bone metastasis in breast cancer (Powell, G. J. Cancer Res (1991) 51:3059-3061; Southby, J. et al. Cancer Res (1990) 50:7710-7716) and has been considered as a possible autocrine factor for some tumors (Burton, P. B. J. et al. Biochem Biophys Res Com (1990) 167:1134-1138; Li, X. et al. Cancer Res (1993) 53:2980-2986).
Thus, the precise role of PTH-rp in mediating the effects of a primary cancer is not well understood. Not only are there additional factors which also participate in the progress of this condition, the of PTH-rp is believed affected by additional factors such as prolactin, flucocrticoids, epidermal growth factor, TGF-.alpha., TGF-.beta., estrogen, "stretch," and even extracellular calcium concentration.
Sato, K. et al. J Bone Min Res (1993) 8:849-860 describe results obtained in a murine model of HHM wherein the affected mice were administered a monoclonal murine antibody obtained from immortalized spleen cells of mice injected with a peptide representing amino acid positions 1-34 of PTH-rp. Passive immunization resulted in decreases in serum calcium concentration in these hypercalcemia nude mice that had been transplanted with human PTH-rp-producing tumors. The authors suggest that if a human counterpart to this antibody could be obtained, it might be used in treatment where malignancy-associated hypercalcemia is due to PTH-rp. In this model, elevated levels of PTH-rp are maintained in the blood which are, evidently, mitigated by the administration of anti-PTH-rp antibodies. The relevance of this model is verified by the finding of Tashjian, A. H. et al. J Exp Med (1964) 119:467 that 15% of 147 hypercalcemia breast cancer patients exhibited no bone metastases.
Hypercalcemia may also be caused by osteolysis of bone through the mediation of osteoclasts (Broadus, A. E. N Engl J Med (1988) 319:556-563). In addition, Mundy, G. R. J Clin Invest (1988) 82:1-6 describes increased osteoclastic bone resorption in areas surrounding breast cancer metastases and breast cancer cells have been shown to resorb bone directed in vitro by Gutierrez, G. E. et al. Ballilere Clin Endocrinol Met (1990) 4:119-138.
Kohno, N. et al. Proc Natl Acad Sci USA (1993) 4:215-220 reported studies performed on immobilized sections of surgically removed breast cancers using an anti-PTH-rp antibody also prepared by immunizing mice with the first 34 amino acids of PTH-rp. The antibody bound to 57% of the tumors; it bound to 83% of the tumors derived from patients who developed skeletal metastases but only 38% in those who either developed lung metastases or no metastases at all. These authors conclude that their results suggest that PTH-rp-positive tumors have an affinity to bone.
The present inventors have developed a model of human breast cell cancer metastasis to bone that results in osteolysis. Nakai, M. et al. Cancer Res (1992) 52:5395-5399; Sasakai, A. et al. J Bone Min Res (1993) 8: (Supplement 1): No. 92. Tumor cells introduced into the left cardiac ventricle of nude mice can be shown to cause osteolytic lesions that can be seen by x-ray examination and that can be confirmed histologically. A375 melanoma cells and the human breast cancer cell line MDA231 have been used in this model. Using this model, it was demonstrated that bone metastasis could be prevented by a synthetic antagonist to laminin.
The present inventors: have also demonstrated that the number of osteolytic lesions is increased by enhancing PTH-rp expression of the MDA231 cells used in the model. Overexpressing clones were obtained by transfecting MDA231 cells with cDNA encoding human prepro PTH-rp; clones having diminished production of this peptide were obtained by transfecting the cell line with an antisense construct. A clone showing elevated expression (100 pM/24 hours) produced 16.3.+-.3.8 lesions radiographically at 3 weeks as compared with the antisense construct which secreted less than 0.3 pM/24 hours which produced only 7.6.+-.0.22 lesions:. No plasma concentrations of PTH-rp were detectable in any of the mice used in these studies; hypercalcemia was minimal and present only in mice harboring the PTH-rp-enhanced MDA231 cells. The present inventors have also shown that PTH-rp concentrations in serum-free media conditioned by MDA231 cells were increased two-fold when the cells were cultured on an extracellular matrix produced by bone cells. These results were reported by Guise, T. A. et al. Breast Cancer Res.trat (1994) 82:79.
It has now been found that antibodies specific for PTH-rp are affective in inhibiting metastasis and ameliorating the effects of malignant cells localized in bone. This provides an effective pharmacological approach to treatment.